The existence of a gating mechanism has long been invoked to explain the vectoriality of proton pumping in bR. Evidence from kinetic experiments suggest this switching occurs in the M intermediate of the photocycle. X-ray, neutron diffraction and electron microscopy results suggest tilting of the F helix and changes in the G helix during the photocycle, which alters the accessibility of the Schiff base to the cytoplasm and may therefore be part of the switching mechanism. Recent work in this laboratory demonstrated changes in the environment of at least two valine backbone carbonyls between the light adapted (LA), Mo and Mn intermediates. Dipolar recoupling experiments indicated that the changes between the LA and Mo states are due to at least two of the five valine residues in helices F and G that are within a helix turn of each other. The difference between the LA and Mn states, however, are not due to any valine carbonyls within 5 A of another valine carbonyl, which rules out all the valines in helices F and G except for valine 167. [unreadable]As the carbonyl of valine 167 is the only valine carbonyl within 3.5 A of an alanine backbone carbonyl, the doubly labeled [1-"C]Ala[l-"C]Val bR will permit determination via dipolar recoupling of any change in the valine 167 carbonyl chemical shift between the LA, Mo and Mn states. Any tilt of the F helix to open then cytoplasmic end of the channel will have the largest effect at this position in the protein. Before using the doubly labeled sample, it is necessary to investigate the [1-"C]Ala labeled bR. Cross-polarization experiments established that at least two alanine carbonyl residues change chemical shift from the LA state to the Mo intermediate and the same change persists into the Mn intermediate. Dipolar recoupling with the MELODRAMA pulse sequence will indicate whether any of these changes are due to next-neighbor alanine residues. If the chemical shift changes are of alanine carbonyls within 3.5 A of other alanine carbonyls, these spins will also be recoupled in the doubly labeled protein. Knowledge of whether or not these alanine carbonyls will be recoupled will pen-nit assignment of the NEWDRAMA spectra of [1_13CIAla[ 1_13CIVal bR and therefore determination of any changes in the valine 167 chemical shift.